Goldenseal (Hydrastis canadensis) 43525850
Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1) CSV JSON

An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 μM and 4.4 μM, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammo- nium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK- OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates.

1 . Berberine Transport by OCT1 (id=NPDI-zv6CYw)

In Vitro Transporter Kinetics Experiment

Transport Activity was detected.  Cutoff used — not specified

Berberine 19012197

  • OCT1 (SLC22A1)

Transfected / siRNA knock-out / injected cells MDCK transfected cells

Results

FIG 5C final data point

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Cell Culture Conditions

1 x 10^8 cm^2

12 well

Cellstar or Thincert

2-4

Assay Conditions

60 min

37 deg C

7.4

1.6 to 48 μM

2 . P-gp Transport of Berberine (id=NPDI-ldcSbA)

In Vitro Transporter Kinetics Experiment

Transport Activity was detected.  Cutoff used — not specified

Berberine 19012197

  • P-gp (ABCB1)

Transfected / siRNA knock-out / injected cells MDCK transfected cells

Results

Fig 6b estimate at 60 min

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Cell Culture Conditions

1 x 10^8 cm^2

12 well

Cellstar or Thincert

2-4

Assay Conditions

60 min

37 deg C

7.4

25 μM

3 . Berberine Transport by OCT2 (id=NPDI-PeLrDg)

In Vitro Transporter Kinetics Experiment

Transport Activity was detected.  Cutoff used — not specified

Berberine 19012197

  • OCT2 (SLC22A2)

Transfected / siRNA knock-out / injected cells MDCK transfected cells

Results

Fig 5d final data point

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

Cell Culture Conditions

1 x 10^8 cm^2

12 well

Cellstar or Thincert

2-4

Assay Conditions

60 min

37 deg C

7.4

1.6 to 48 μM